The major goal of our lab has been to utilize a platform technology of retrovirally-delivered gene therapy for tolerance induction in autoimmune diseases, with a focus on Type 1 diabetes. These studies are based on the tolerogenicity of Ig carriers and B-cell presentation of processed T-cell epitopes. During the last decade, we have engineered multiple epitope-containing polypeptides in frame on a murine lgG1 heavy chain scaffold, and expressed them in B cells to achieve the induction and long-term maintenance of immune tolerance through continuous in vivo presentation of relevant epitopes. Data in several experimental autoimmune models are highly promising in that significant clinical efficacy has been achieved. Thus, processing and presentation of Ig fusion proteins by B cells can both prevent and reverse autoimmune responsiveness in uveitis and EAE. Importantly, we have found that expression of full length GAD65 (GAD- Ig) and insulin B9-23-lg (or pro-insulin-lg) in NOD B cells significantly delayed the onset of diabetes in female NOD mice even when treatment was started at 7-12 weeks of age (i.e., in mice with peri-insulitis). Our hypothesis is that the fusion GAD-lg is processed by NOD B cells (both male and female) for tolerogenic presentation of both major and minor GAD epitopes, and leads to the generation of T regulatory cells. Herein, we will determine the optimal conditions for B-cell activation and the mechanism of this "tolerance". We also will directly establish whether this gene therapeutic approach targets CD4 and CDS T cells or both in terms of cytokine production to multiple epitopes, as well as their ability to transfer diabetes to NOD-scid recipients. We will follow the fate of target T cells using TCR transgenic lines recognizing CD4 or CDS epitopes. Moreover, we will examine the role and specificity of regulatory T cells may be involved in both the induction and maintenance of tolerance in direct ablation and cell mixing experiments, as well as determine the role of suppressive cytokines in tolerance. Importantly for future therapy, we also wish to provide proof of principle for the efficacy of this system in an islet transplantation model. Our aims are: 1) To optimize and determine the targets of B-cell delivered gene therapy for tolerance, 2) To identify the cellular targets and mechanism of B-cell delivered gene therapy. 3) To examine the fate of pathogenic T cells and the role of regulatory T cells after B-cell delivered gene therapy, and 4) To establish the efficacy of B-cell delivered gene therapy in an islet transplant model. The accomplishment of the aims will establish the role of regulatory T cells, as well as the cellular targets for B-cell delivered gene therapy for tolerance. Our goal is to modulate pathogenic responses to GAD, for example, as a prelude to applying this protocol as a complementary